AlphaLISA SureFire Ultra Human Total MERTK Detection Kit, 100 Assay Points

Total-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.

Validation of MERTK Total in cells treated with Gas6 protein

PANC-1 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Gas6 protein for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, the Gas6 protein triggered a dose-dependent increase in levels of Phospho (Tyr749) while MERTK Total remained unchanged.

Validation of MERTK Total in H₂O₂ treated cells

Caco-2 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of H₂O₂ for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MERTK Phospho (Tyr749) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, H₂O₂ treatment triggered a dose-dependent increase in levels of Phospho (Tyr749) while MERTK Total remained unchanged.

Specificity of MERTK Total assay

Specificity of the Total MERTK assay was assessed by assaying recombinant MERTK, AXL and Tyro3 proteins. Dilutions of recombinant MERTK, AXL, and Tyro3 proteins were prepared in Lysis Buffer. MERTK Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Total MERTK assay was specific only to MERTK recombinant protein with no signal detected with AXL and Tyro3 recombinant proteins. This result demonstrates the specificity of the Total MERTK SureFire Ultra assay as these three proteins share up to 46% sequence identity.

Validation of MERTK Total assay in various cell line

Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL of Lysis Buffer.
Suspension cells (Daudi, Ramos) were seeded at 100,000 cells/well (200 µL/well) in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 100 µL of Lysis Buffer. Suspension cell lysates were further diluted 1 in 2.5 with Lysis Buffer.

MERTK Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT.  Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Total MERTK expression is dependent upon cell type. As expected, a very high level of expression was detected in HepG2 and PANC-1 cell lines, while signal was undetectable in HeLa cells.